EpiGenie Guide: Rna Immunoprecipitation (RIP) and Beyond
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EpiGenie Guide:
Rna Immunoprecipitation (RIP)
And Beyond
RNA in a Bind
RNA binding proteins (RBPs) play
important roles in many cellular
and disease processes, together
with their DNA binding brethren,
they compromise almost one
quarter of annotated human
genes. RBPs can act in a highly
sequence-specific context and are
involved in RNA splicing, editing,
translational regulation and RNA
degradation. The tools used to
understand when and where
these proteins bind and interact
with RNAs in the cell have lagged
behind those for protein-DNA
interactions, although they are
rapidly gaining ground. This guide
In this guide… will describe some of the more
• Rna-Immunoprecipitation recently used tools to examine
• RIP-ChIP protein-RNA binding including the
• CLIP And Its Variants variants of RIP and CLIP and their
• ChIPing For Rna combination with next-generation
• RIP & CLIP Analysis sequencing (NGS).
RNA- This material is then usually sonicated to While high-density arrays and high
produce fragments of 300-500bp and throughput sequencing unleashed
immunoprecipitation DNAse treatment removes DNA before the coverage constraints, it became
RNA Binding Protein Immuno immunoprecipitation with an antibody clear in these early studies that the
precipitation allows users to analyze against the protein of interest enriches formaldehyde cross-linking had a few
protein-RNA binding in the same the bound RNA molecules. Early RIP hiccups in the approach that could
way as the more familiar Chromatin- studies analyzed only a few loci; the lead to challenges downstream. Robert
immunoprecipitation (ChIP) is used RNA fragments were used in reverse- Darnell detailed some of his team’s
to study protein-DNA binding. transction PCR to identify differential experiences with RIP and issues with
Immunoprecipitation with an antibody binding of RNAs to proteins one locus signal to noise ratios in their attempts
against a protein of interest enriches at a time, with gel mobility shift assays to investigate the RNA targets of the
for RNA molecules actively bound to or using clone library screening for a fragile-X mental retardation protein,
the target protein. ”The value in RIP more comprehensive analysis, but the FMRP. They found that the approach
is in being able to isolate lysates that downstream analysis approach quickly often lead to co-purification of
are relatively ribosomal RNA free for ramped up. non-target molecule RNAs. But if
the purposes of profiling protein-RNA formaldehyde cross-linking wasn’t
interactions. This method is useful Stepping Up used, then RNA and RBPs could easily
for both cytoplasmic and nuclear Coverage: RIP-ChIP dissociate and re-associate in vitro.
protein-RNA interactions from primary
or cultured cells,” shared Dr. John and RIP-Seq Both of these issues required researchers
to use carefully controlled and designed
Rosenfeld, R&D Group Leader at EMD Although the early RNA experiments to make the most of RIP.
Millipore, a producer of optimized RIP immunoprecipitation studies led to Fortunately, advances in cross-linking
kits and reagents. many interesting discovers, researchers were on the horizon.
needed higher coverage analysis
Initially RIP studies used a native
immunoprecipitation without any form
gain a richer understanding of RNA Getting into the
regulatory networks. This led researchers
of cross-linking. The conditions required
to develop assays that allowed the Specifics with CLIP:
had three significant issues:
interrogation of bound RNA using Cross-Linking
• RNAs bound to protein other than the microarrays (RIP-ChIP) and subsequently
target can potentially co-precipitated RNA sequencing (RIP-Seq). Immunoprecipitation
• Reaction conditions preserved only a UV cross-linking and
Similar to the transition seen with ChIP,
subset of cellular interactions immunoprecipitation (CLIP) was
these powerful downstream analysis
developed by Jernej Ule at Howard
• In vitro artifactual interactions may be techniques uncovered diverse and
Hughes Medical Institute while in
detected formed during the process extensive protein-RNA interactions.
Robert Darnell’s group. CLIP makes use
Daniel Hogan and colleagues provided
To improve the stability of the of the photoreactivity of pyrimidines
a glimpse into the RNA interactome in
interactions, cells or tissue can be treated and specific amino acids to produce
yeast back in 2008 when they found a
with formaldehyde that generates irreversible covalent crosslinks between
fraction of the predicted RNA binding
protein-protein, protein-DNA, and RBP’s and their bound RNA’s, while
proteins bound anywhere from a few to
protein-RNA cross-links. completely avoiding protein:protein
hundreds of target mRNA.
cross-linking.
CLIP can be performed on live cells CLIP-Seq On PAR-CLIP:
preserving a snapshot of a specific in
vivo environment and is done rapidly There haven’t been many applications Photoactivable
at 4C using UV irradiation. It has that high throughput sequencing
couldn’t improve. CLIP has been no
Ribonucleotid
been suggested that the rapidity of
treatment allows very little biological exception. Next-generation sequencing Enhanced CLIP
reaction to occur, which can confound of CLIP was first demonstrated as high- To address some of the issues
experimental results, however it is throughput sequencing of RNA isolated experienced with the first generation
worth considering the impact of DNA by crosslinking immunoprecipitation CLIP protocol, Hafner and colleagues
damage responses if the process is (HITS-CLIP) by Robert Darnell’s group in developed, PAR-CLIP (Photoactivatable-
completed on cells that will be kept 2008 using the Roche-454 sequencing Ribonucleoside-Enhanced Crosslinking
living after treatment. platform. and Immunoprecipitation), which
Their analysis of the Nova splicing modified CLIP further by incorporating
With CLIP, it’s possible to use a
factor showed how reproducible the photoreactive nucleotide analogs,
rigorous purification, which can be
technique was. They created genome- 4-thiouridine (4-SU) and 6-thioguanosine
helpful in removing artifacts. The
wide maps of Nova:RNA binding, and (6-SG), into RNA molecules during
fragments of RNA recovered after
demonstrated that Nova plays a role in transcription. UV irradiation at 365nm
immunoprecipitation can then be
alternative polyadenylation in the brain. was then used to induce a very efficient
cloned, amplified by PCR and, in the
crosslinking of the now photoreactive
original paper were Sanger-sequenced. The term CLIP-seq was first used by cellular RNAs and the proteins to which
But some experts suggest to proceed Jeremey Sanford and colleagues from they were bound. A nice upside to the
with caution. UCSC in 2009. They used CLIP-seq PAR-CLIP approach is that it’s possible to
to determine the RNA binding precisely map the position of crosslinking,
“Like the chromatin cross- specificity of SFRS1, a regulator of which helps resolve some of the analysis
linked based methods, there pre-mRNA splicing. They showed that sometimes seen with CLIP. This benefit
can be higher background many different RNA molecules were comes from the fact that crosslinking
bound by SFRS1, not just mRNA’s but the 4-SU analog results in thymidine to
when dealing with these
also micro-, non-coding- and other cytidine transitions, where as crosslinking
samples, and acrylamide RNA species; all of which shared a the 6-SG analog results in guanosine to
isolation of enriched protein- consensus-binding motif. This binding adenosine mutations. These mutations
RNA is a key step in this motif was used to screen the Human make it easier to separate the crosslinked
process to eliminate both Gene Mutation Database for mutations RNA from other, more abundant cellular
disrupting SFRS1 binding where they RNA. The downside? PAR-CLIP is currently
protein partners in a complex,
found nearly 200 mutations in over 80 limited to in vitro cell culture studies
as well as extraneous RNA genes suggesting a role for aberrant and the analogs used are not perfectly
products.” splicing in Human disease. tolerated by cells.
Dr. John Rosenfeld
But, like many methods CLIP isn’t
R&D Group Leader, EMD Millipore
infallible. Markus Hafner and colleagues
review some of the issues with CLIP.
The wavelength of crosslinking in CLIP
(UV 254 nm) isn’t always efficient and
the mapping the precise location of
the cross-linked material isn’t easy.
This can make separating the relevant
interactions from some of the carryover
RNA difficult.
iCLIP: Individual Stepping into performing ChIP to examine RNA, standard
ChIP protocols can be used with minor
Nucleotide Resolution Chromatin: ChIP adjustments such as using RNAse free
CLIP As we’ve outlined here, there are number of reagents, modified binding and wash
ways to pin down protein-RNA interactions, buffers, the inclusion of two DNAse I
No, Apple hasn’t sprung into the RNA
but sometimes it pays to take the reverse treatment steps in the presence of Mg2+
analysis market…yet. iCLIP (individual-
approach and head right to the context… and Ca2+ (one before the IP step and
nucleotide resolution CLIP), is another
like chromatin for instance. one after the purification) to decrease
interesting innovation that improves
background. Following elution RNA is
the efficiency of CLIP by replacing an So how does ChIP work when it’s purified using Trizol, reverse transcribed and
inefficient RNA ligation step with a more RNA you’re after? Surprisingly, not that analyzed by PCR; the inclusion of a no RT
efficient cDNA circularization and at the much different as Kelly explains, “ When control will measure DNA contamination.”
same time adds a random barcode to the
DNA adaptor. Sometimes cDNA synthesis
from CLIP libraries can truncate at the “Several techniques are available to identify protein-RNA
crosslinked loci. This can be a problem, but interactions, however a direct role for RNA in chromatin
iCLIP uses it to its advantage; by capturing regulation through microRNAs and lncRNAs, is becoming
these truncated cDNAs, it is possible to increasingly more apparent. Traditionally, chromatin
map the cross-linked contacts with single
nucleotide precision. iCLIP is a useful
immunoprecipitation is preformed using antibodies to
CLIP variant, but like other approaches, histones or DNA binding proteins followed by identification
it comes with some fine print. There are of the associated DNA regions, however it can be adapted
a number of enzymatic and purification to examine RNAs that are associated with chromatin. The
steps to work through, which means there use of fixed material to examine chromatin-RNA interactions
are plenty of opportunities for things to
go wrong, so you’ll want to use a host of
stabilizes the dynamic chromatin environment which can
QC/checkpoints. The authors make some increase sensitivity over methods using unfixed material.”
good suggestions in their discussion that Dr. Terry Kelly, Active Motif R&D Manager.
should serve as a useful guide.
Summary of the Cross-link protein to RNA & DNA in Lyse cells and shear both RNA Treat with DNase I Add Protein G Magnetic Beads &
CLIPings
living cells with formaldehyde & DNA by Sonication Shearing to remove most DNA primary antibody of interest
Although the early CLIP-seq studies allowed
the first comprehensive genome-wide
maps of protein-RNA interaction to be
generated, they weren’t flawless. Several
deficiencies in these methods were
observed by the authors including the Analyze purified RNA
using end point or
need for significantly higher sequencing real-time RT-PCT
depth and the impact of PCR artifacts on
Treat with DNase I again Elute RNA from Magnetic Beads, Immunoprecipitate antibody-bound
quantitation of individual transcripts. That to remove any residual DNA reverse protein/RNA cross-links,
then extract the RNA
protein/RNA complexes using
Magnetic Beads and magnet
said, the majority of CLIP-seq projects Flow chart of the RNA ChIP-IT™ process.
have been run on Illumina sequencing Live cells are fixed using formaldehyde, which cross-links and preserves protein/RNA interactions
(as well as protein/DNA interactions). The RNA/DNA is then sheared into small, uniform fragments
instruments that today can generate 3B using sonication and, after a DNase treatment to remove residual DNA, specific protein/RNA complexes
are immunoprecipitated using an antibody directed against the RNA-binding protein of interest.
reads per run, allowing 100’s of samples to Following immunoprecipitation, cross-linking is reversed, the RNA is extracted then DNase I treated again
(to remove residual DNA). The RNA is then analyzed by end point or real-time RT-PCR to determine which
be analyzed in just a few days. We expect RNA fragments were bound by the protein of interest.
continued innovation in this exciting area.John Rosenfeld adds, “There are Validating RIP co-transfected with either a cDNA
emerging methods that involve expression vector or a siRNA to a RNA-
isolating nuclei for the specific purpose Results and binding protein of interest to measure
of isolating non-coding protein-RNA Measuring Functional the activity of the 3’UTR in the absence
interactions from chromatin. Nuclear or presence of the RNA-binding protein.
RIP can achieve this without the Consequences
Likewise, the 3’UTR reporter vector can
use of crosslinking agents such as “When a protein-RNA interaction is
be co-transfected with a miRNA mimic
formaldehyde, which can be useful detected in a RIP experiment, it is often
or inhibitor to measure the activity of
for confirming results obtained from desirable to validate the interaction
the 3’UTR in the presence or absence of
formaldehyde crosslinking experiments.” and characterize the functional
a particular miRNA (see figure below). As
consequences of that interaction. Since
“The sample type for these protocols a final confirmation, the 3’UTR sequence
many RNA-binding proteins including
can either be a native, nuclear RIP lysate can be mutated in the reporter vector to
the miRNA-RISC complex interact
where DNA components are degraded, either remove a miRNA seed sequence
with the 3’UTRs of protein coding
or can be prepared chromatin, which is or disrupt a secondary structure
transcripts, 3’UTR reporter constructs
more like a ChIP reaction in the presence necessary for protein binding.
are a popular way to assess the function
of RNA inhibitors and with DNAse
of an RNA:protein interaction,” shared
treatment to remove contaminating
Dr. Nathan Trinklein, SwitchGear
DNA from chromatin. These methods
Genomics (an Active Motif company).
are complementary, as crosslinking
chromatin can elucidate interactions, In this method a 3’UTR is cloned
which may not be high affinity or downstream of a reporter gene on
bona fide binding events. Generally, a plasmid vector. When this vector is
RIP, Nuclear RIP and Cross-linked IP transfected into a cell, it creates a hybrid
can utilize qRT-PCR as a readout when transcript that contains the reporter
a target species is known, or can be gene fused to the 3’UTR of interest.
applied to library construction for The 3’UTR reporter construct can be
ncRNA-seq discovery experiments,”
stated Rosenfeld.
More Tips and Protocol Guidance
For more grit on these methods and other useful tools check out these great reviews and publications:
HITS-CLIP: panoramic views of protein-RNA regulation in living cells
This is an amazing review that covers, CLIP and CLIP-seq and provides tons of insightful commentary on the methods,
their utility, and future areas for improvement.
PAR-CLIP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins
This nice pub goes into good depth about some of the issues you may run into with standard CLIP-seq, and introduces the
ehancements and utility of PAR-CLIP.
iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution
Here’s a nice introduction to iCLIP complete with a some visual razzle dazzle courtesy of Jove.
RIPSeeker: a statistical package for identifying protein-associated transcripts from RIP-seq experiments
This nice paper introduces one of the only software packages out there for analyzing RIP-seq data.Product Catalog Number EMD Millipore RNA-Binding Protein Immunoprecipitation Kit Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit 17-700 EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit 17-701 Magna RIP™ Quad RNA-Binding Protein Immunoprecipitation Kit 17-704 Active Motif RNA ChIP Kits RNA ChIP-IT® 53024 RNA ChIP-IT® Control Kit - Human 53025 Reporter Systems for Protein-RNA Interactions LightSwitch™ Promoter and 3´UTR Reporter Collections Variable LightSwitch™ Luciferase Assay Kit 32031 LightSwitch™ Luciferase Assay Kit 32032 Empty LightSwitch™ Reporter Vectors Variable www.epigenie.com This item is intended for research use only. Not for use in diagnostic procedures. Information, descriptions, and specifications in this publication are subject to change without notice.
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